Antibodies to aripiprazole and use thereof

ABSTRACT

Disclosed is an antibody which binds to aripiprazole, which can be used to detect aripiprazole in a sample such as in a competitive immunoassay method. The antibody can be used in a lateral flow assay device for point-of-care detection of aripiprazole, including multiplex detection of aripiprazole, olanzapine, quetiapine, and risperidone in a single lateral flow assay device.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/971,448, filed Aug. 20, 2013, published on Feb. 27, 2014, as US2014/0057300, and claims the benefit of U.S. Provisional Application No.61/691,522, filed Aug. 21, 2012, all of which are hereby incorporated byreference in their entireties.

FIELD OF THE INVENTION

The present invention relates to the field of immunoassays, and inparticular to antibodies that bind to aripiprazole which can be used inimmunoassays for detection of aripiprazole.

BACKGROUND

Schizophrenia is a chronic and debilitating psychiatric disorderaffecting approximately 0.45-1% of the world's population (van Os, J.;Kapur, S. “Schizophrenia” Lancet 2009, 374, 635-645). The principalgoals of treatment are to achieve sustained remission from psychoticsymptoms, reduce the risk and consequences of relapse, and improvepatient functioning and overall quality of life. While many patientswith schizophrenia are able to achieve symptom stability with theavailable antipsychotic medications, poor adherence to medication is acommon reason for relapse with daily administered oral medications.Several studies (Abdel-Baki, A.; Ouellet-Plamondon, C.; Malla, A.“Pharmacotherapy Challenges in Patients with First-Episode Psychosis”Journal of Affective Disorders 2012, 138, S3-S14) investigating theoutcomes of non-compliance have shown that patients with schizophreniawho do not take their medication as prescribed have higher rates ofrelapse, hospital admission and suicide as well as increased mortality.It is estimated that 40 to 75% of patients with schizophrenia havedifficulty adhering to a daily oral treatment regimen (Lieberman, J. A.;Stroup, T. S.; McEvoy, J. P.; Swartz, M. S.; Rosenheck, R. A.; Perkins,D. O.; Keefe, R. S. E.; Davis, S. M.; Davis, C. E.; Lebowitz, B. D.;Severe, J.; Hsiao, J. K. “Effectiveness of Antipyschotic Drugs inPatients with Chronic Schizophrenia” New England Journal of Medicine2005, 353(12), 1209-1223).

Therapeutic drug monitoring (TDM) is the quantification of serum orplasma concentrations of drugs, including anti-psychotic drugs, fortreatment monitoring and optimization. Such monitoring permits, forexample, the identification of patients that are not adhering to theirmedication regimen, that are not achieving therapeutic doses, that arenon-responsive at therapeutic doses, that have suboptimal tolerability,that have pharmacokinetic drug-drug interactions, or that have abnormalmetabolism resulting in inappropriate plasma concentrations.Considerable individual variability exists in the patient's ability toabsorb, distribute, metabolize, and excrete anti-psychotic drugs. Suchdifferences can be caused by concurrent disease, age, concomitantmedication or genetic peculiarities. Different drug formulations canalso influence the metabolism of anti-psychotic drugs. TDM permits doseoptimization for individual patients, improving therapeutic andfunctional outcomes. TDM further permits a prescribing clinician toensure compliance with prescribed dosages and achievement of effectiveserum concentrations.

To date, methods for determining the levels of serum or plasmaconcentrations of anti-psychotic drugs involve the use of liquidchromatography (LC) with UV or mass spectrometry detection, andradioimmunoassays (see, for example, Woestenborghs et al., 1990 “On theselectivity of some recently developed RIA's” in Methodological Surveysin Biochemistry and Analysis 20:241-246. Analysis of Drugs andMetabolites, Including Anti-infective Agents; Heykants et al., 1994 “ThePharmacokinetics of Risperidone in Humans: A Summary”, J Clin Psychiatry55/5, suppl: 13-17; Huang et al., 1993 “Pharmacokinetics of the novelanti-psychotic agent risperidone and the prolactin response in healthysubjects”, Clin Pharmacol Ther 54:257-268). Radioimmunoassays detect oneor both of risperidone and paliperidone. Salamone et al. in U.S. Pat.No. 8,088,594 disclose a competitive immunoassay for risperidone usingantibodies that detect both risperidone and paliperidone but notpharmacologically inactive metabolites. The antibodies used in thecompetitive immunoassay are developed against a particular immunogen. IDLabs Inc. (London, Ontario, Canada) markets an ELISA for olanzapine,another anti-psychotic drug, which also utilizes a competitive format.The Instructions For Use indicate that the assay is designed forscreening purposes and intended for forensic or research use, and isspecifically not intended for therapeutic use. The Instructionsrecommend that all positive samples should be confirmed with gaschromatography/mass spectrometry (GC-MS), and indicate that the antibodyused detects olanzapine and clozapine (see ID Labs Inc., “InstructionsFor Use Data Sheet IDEL-F083”, Rev. Date Aug. 8, 2011). Some of thesemethods, namely HPLC and GC/MS, can be expensive and labor-intensive,and are generally only performed in large or specialty labs having theappropriate equipment.

A need exists for other methods for determining the levels ofanti-psychotic drugs, particularly methods that can be performed in aprescribing clinician's office (where the treatment for an individualpatient can be adjusted accordingly in a much more timely manner) and inother medical settings lacking LC or GC/MS equipment or requiring rapidtest results.

SUMMARY OF THE INVENTION

The present invention is directed to an isolated antibody or a bindingfragment thereof, which binds to aripiprazole and which: (i) is anantibody selected from the group consisting of: a) an isolated antibodyor a fragment thereof comprising a light chain variable regioncomprising the amino acid sequence of SEQ ID NO:43, SEQ ID NO:47, SEQ IDNO:51 or SEQ ID NO:55; b) an isolated antibody or a fragment thereofcomprising a heavy chain variable region comprising the amino acidsequence of SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52 or SEQ ID NO:56; c)an isolated antibody or a fragment thereof comprising a light chainvariable region having the amino acid sequence of SEQ ID NO:43 and aheavy chain variable region having the amino acid sequence of SEQ IDNO:44; d) an isolated antibody or a fragment thereof comprising a lightchain variable region having the amino acid sequence of SEQ ID NO:47 anda heavy chain variable region having the amino acid sequence of SEQ IDNO:48; e) an isolated antibody or a fragment thereof comprising a lightchain variable region having the amino acid sequence of SEQ ID NO:51 anda heavy chain variable region having the amino acid sequence of SEQ IDNO:52; f) an isolated antibody or a fragment thereof comprising a lightchain variable region having the amino acid sequence of SEQ ID NO:55 anda heavy chain variable region having the amino acid sequence of SEQ IDNO:56; or (ii) competes for an epitope which is the same as an epitopebound by the antibody of (i).

The antibodies of the subject invention can be provided in assay kitsand assay devices, with a presently preferred device being a lateralflow assay device which provides for point-of-care analysis.

The invention further provides a method of detecting aripiprazole in asample. The method comprises: (i) contacting a sample with an antibodyaccording to the subject invention which is labeled with a detectablemarker, wherein the labeled antibody and aripiprazole present in thesample form a labeled complex; and (ii) detecting the labeled complex soas to detect aripiprazole in the sample.

Further provided is a competitive immunoassay method for detectingaripiprazole in a sample. The method comprises: (i) contacting a samplewith an antibody according to the subject invention, and witharipiprazole or a competitive binding partner of aripiprazole, whereinone of the antibody and the aripiprazole or competitive binding partnerthereof is labeled with a detectable marker, and wherein samplearipiprazole competes with the aripiprazole or competitive bindingpartner thereof for binding to the antibody; and (ii) detecting thelabel so as to detect sample aripiprazole.

Further objects, features and advantages of the present invention willbe apparent to those skilled in the art from detailed consideration ofthe preferred embodiments that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows Competitive ELISA results generated with hybridoma 3C1;

FIG. 2 shows Competitive ELISA results generated with hybridoma 3D7;

FIG. 3 shows the competitive immunoassay format used on a lateral flowassay device;

FIG. 4 shows the chip design of a lateral flow assay device according tothe subject invention;

FIG. 5 shows a typical dose response curve for an aripiprazole positivecontrol generated with antibody 5C7 and a labeled aripiprazolecompetitive binding partner;

FIG. 6 shows a typical dose response curve for an olanzapine positivecontrol generated with antibody 4G9-1 and a labeled olanzapinecompetitive binding partner;

FIG. 7 shows a typical dose response curve for a quetiapine positivecontrol generated with antibody 11 and a labeled quetiapine competitivebinding partner;

FIG. 8 shows a typical dose response curve for a risperidone positivecontrol generated with antibody 5-9 and a labeled risperidonecompetitive binding partner;

FIG. 9 shows a typical dose response curve for a sample containingaripiprazole generated with aripiprazole antibody 5C7 in the presence oflabeled aripiprazole competitive binding partner, with no dose responsecurve for olanzapine, quetiapine, or risperidone in the presence of alabeled competitive binding partner for each;

FIG. 10 shows a typical dose response curve for a sample containingolanzapine generated with olanzapine antibody 4G9-1 in the presence of alabeled olanzapine competitive binding partner, with no dose responsecurve for aripiprazole, quetiapine, or risperidone in the presence of alabeled competitive binding partner for each;

FIG. 11 shows a typical dose response curve for a sample containingquetiapine generated with quetiapine antibody 11 in the presence of alabeled quetiapine competitive binding partner, with no dose responsecurve for aripiprazole, olanzapine, or risperidone in the presence of alabeled competitive binding partner for each;

FIG. 12 shows a typical dose response curve for a sample containingrisperidone generated with risperidone antibody 5-9 in the presence of alabeled risperidone competitive binding partner, with no dose responsecurve for aripiprazole, olanzapine, or quetiapine in the presence of alabeled competitive binding partner for each;

FIG. 13 shows a typical dose response curve for a sample containingaripiprazole generated with aripiprazole antibody 5C7 in the presence ofa labeled aripiprazole competitive binding partner, with no doseresponse curve for olanzapine, quetiapine, or risperidone in thepresence of antibody and labeled competitive binding partner for each;

FIG. 14 shows a typical dose response curve for a sample containingolanzapine generated with olanzapine antibody 4G9-1 in the presence of alabeled olanzapine competitive binding partner, with no dose responsecurve for aripiprazole, quetiapine, or risperidone in the presence ofantibody and labeled competitive binding partner for each;

FIG. 15 shows a typical dose response curve for a sample containingquetiapine generated with quetiapine antibody 11 in the presence oflabeled quetiapine competitive binding partner, with no dose responsecurve for aripiprazole, olanzapine, or risperidone in the presence ofantibody and labeled competitive binding partner for each;

FIG. 16 shows a typical dose response curve for a sample containingrisperidone generated with risperidone antibody 5-9 in the presence of alabeled risperidone competitive binding partner, with no dose responsecurve for aripiprazole, olanzapine, or quetiapine in the presence ofantibody and labeled competitive binding partner for each;

FIG. 17 shows a comparison of the aripiprazole dose response curvegenerated as a positive control to the aripiprazole dose response curvegenerated in the multiplex format;

FIG. 18 shows a comparison of the olanzapine dose response curvegenerated as a positive control to the olanzapine dose response curvegenerated in the multiplex format;

FIG. 19 shows a comparison of the quetiapine dose response curvegenerated as a positive control to the quetiapine dose response curvegenerated in the multiplex format; and

FIG. 20 shows a comparison of the risperidone dose response curvegenerated as a positive control to the risperidone dose response curvegenerated in the multiplex format.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotide or amino acid sequences: “referencesequence”, “comparison window”, “sequence identity”, “percentage ofsequence identity”, “substantial identity”, “similarity”, and“homologous”. A “reference sequence” is a defined sequence used as abasis for a sequence comparison; a reference sequence may be a subset ofa larger sequence, for example, a segment of a full length cDNA or genesequence given in a sequence listing or may comprise a complete cDNA orgene sequence; a reference sequence may comprise a segment of a completeamino acid sequence encoding a protein as given in a sequence listing ormay comprise a complete amino acid sequence encoding a protein.Generally, a reference sequence is at least 18 nucleotides or 6 aminoacids in length, frequently at least 24 nucleotides or 8 amino acids inlength, and often at least 48 nucleotides or 16 amino acids in length.Since two polynucleotide or amino acid sequences may each (1) comprise asequence (i.e., a portion of the complete nucleotide or amino acidsequence) that is similar between the two molecules, and (2) may furthercomprise a sequence that is divergent between the two polynucleotide oramino acid sequences, sequence comparisons between two (or more)molecules are typically performed by comparing sequences of the twomolecules over a “comparison window” to identify and compare localregions of sequence similarity. A “comparison window”, as used herein,refers to a conceptual segment of at least 18 contiguous nucleotidepositions or 6 amino acids wherein the polynucleotide sequence or aminoacid sequence may be compared to a reference sequence of at least 18contiguous nucleotides or 6 amino acids and wherein the portion of thepolynucleotide sequence or amino acid sequence in the comparison windowmay comprise additions, deletions, substitutions, and the like (i.e.,gaps) of 20 percent or less as compared to the reference sequence (whichdoes not comprise additions or deletions) for optimal alignment of thetwo sequences. Optimal alignment of sequences for aligning a comparisonwindow may be conducted by the local homology algorithm of Smith andWaterman, Adv. Appl. Math 2:482 (1981), by the homology alignmentalgorithm of Needlemen and Wunsch, J. Mol. Biol. 48:443 (1970), by thesearch for similarity method of Pearson and Lipman, Proc. Natl. Acad.Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package Release 7.0 (Genetics Computer Group, 575 Science Dr.,Madison, Wis.), Geneworks or MacVector software packages), or byinspection, and the best alignment (i.e., resulting in the highestpercentage of identity over the comparison window) generated by thevarious methods is selected.

The term “sequence identity” means that two polynucleotide or amino acidsequences are identical (i.e., on a nucleotide-by-nucleotide or aminoacid residue-by-residue basis) over the comparison window. The term“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, or U) or amino acid residue occurs in both sequences toyield the number of matched positions, dividing the number of matchedpositions by the total number of positions in the comparison window(i.e., the window size), and multiplying the result by 100 to yield thepercentage of sequence identity. The term “substantial identity” as usedherein denotes a characteristic of a polynucleotide or amino acidsequence, wherein the polynucleotide or amino acid sequence comprises asequence that has at least 85 percent sequence identity, preferably atleast 90 to 95 percent sequence identity, more usually at least 99percent sequence identity as compared to a reference sequence over acomparison window of at least 18 nucleotide (6 amino acid) positions,frequently over a window of at least 24-48 nucleotide (8-16 amino acid)positions, wherein the percentage of sequence identity is calculated bycomparing the reference sequence to the sequence which may includedeletions or additions which total 20 percent or less of the referencesequence over the comparison window. The reference sequence may be asubset of a larger sequence. The term “similarity”, when used todescribe a polypeptide, is determined by comparing the amino acidsequence and the conserved amino acid substitutions of one polypeptideto the sequence of a second polypeptide. The term “homologous”, whenused to describe a polynucleotide, indicates that two polynucleotides,or designated sequences thereof, when optimally aligned and compared,are identical, with appropriate nucleotide insertions or deletions, inat least 70% of the nucleotides, usually from about 75% to 99%, and moreprefereably at least about 98% to 99% of the nucleotides.

A “label,” “detector molecule,” “reporter” or “detectable marker” asused herein is any molecule which produces, or can be induced toproduce, a detectable signal. The label can be conjugated to an analyte,immunogen, antibody, or to another molecule such as a receptor or amolecule that can bind to a receptor such as a ligand, particularly ahapten or antibody. A label can be attached directly or indirectly bymeans of a linking or bridging moiety. Non-limiting examples of labelsinclude radioactive isotopes (e.g., ¹²⁵I), enzymes (e.g.β-galactosidase, peroxidase), enzyme fragments, enzyme substrates,enzyme inhibitors, coenzymes, catalysts, fluorophores (e.g., rhodamine,fluorescein isothiocyanate or FITC, or Dylight 649), dyes,chemiluminescers and luminescers (e.g., dioxetanes, luciferin), orsensitizers.

The invention provides an isolated antibody which binds to aripiprazole.The invention further provides an assay kit and an assay devicecomprising the antibody. Further provided is a method of detectingaripiprazole in a sample, including a competitive immunoassay method.

In one embodiment, the present invention is directed to an isolatedantibody or a binding fragment thereof, which binds to aripiprazole andwhich: (i) is an antibody selected from the group consisting of: a) anisolated antibody or a fragment thereof comprising a light chainvariable region comprising the amino acid sequence of SEQ ID NO:43, SEQID NO:47, SEQ ID NO:51 or SEQ ID NO:55; b) an isolated antibody or afragment thereof comprising a heavy chain variable region comprising theamino acid sequence of SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52 or SEQID NO:56; c) an isolated antibody or a fragment thereof comprising alight chain variable region having the amino acid sequence of SEQ IDNO:43 and a heavy chain variable region having the amino acid sequenceof SEQ ID NO:44; d) an isolated antibody or a fragment thereofcomprising a light chain variable region having the amino acid sequenceof SEQ ID NO:47 and a heavy chain variable region having the amino acidsequence of SEQ ID NO:48; e) an isolated antibody or a fragment thereofcomprising a light chain variable region having the amino acid sequenceof SEQ ID NO:51 and a heavy chain variable region having the amino acidsequence of SEQ ID NO:52; f) an isolated antibody or a fragment thereofcomprising a light chain variable region having the amino acid sequenceof SEQ ID NO:55 and a heavy chain variable region having the amino acidsequence of SEQ ID NO:56; or (ii) competes for an epitope which is thesame as an epitope bound by the antibody of (i).

In a further embodiment, the present invention is directed to anisolated antibody or a binding fragment thereof, which binds toaripiprazole and which comprises a light chain variable regioncomprising an amino acid sequence having at least 80% sequence identitywith SEQ ID NO:43, SEQ ID NO:47, SEQ ID NO:51 or SEQ ID NO:55.

In a further embodiment, the present invention is directed to anisolated antibody or a binding fragment thereof, which binds toaripiprazole and which comprises a heavy chain variable regioncomprising an amino acid sequence having at least 80% sequence identitywith SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52 or SEQ ID NO:56.

Presently preferred embodiments of the antibody of the subject inventionare: an antibody which comprises a light chain variable region havingthe amino acid sequence SEQ ID NO:43 and a heavy chain variable regionhaving the amino acid sequence SEQ ID NO:44; an antibody which comprisesa light chain variable region having the amino acid sequence SEQ IDNO:47 and a heavy chain variable region having the amino acid sequenceSEQ ID NO:48; an antibody which comprises a light chain variable regionhaving the amino acid sequence SEQ ID NO:51 and a heavy chain variableregion having the amino acid sequence SEQ ID NO:52; an antibody whichcomprises a light chain variable region having the amino acid sequenceSEQ ID NO:55 and a heavy chain variable region having the amino acidsequence SEQ ID NO:56.

Additional presently preferred embodiments of the antibody of thesubject invention are: 1) an antibody which comprises a light chain CDR1sequence comprising amino acid residues 46 to 55 of SEQ ID NO:43, alight chain CDR2 sequence comprising amino acid residues 71 to 77 of SEQID NO:43, a light chain CDR3 sequence comprising amino acid residues 110to 118 of SEQ ID NO:43, a heavy chain CDR1 sequence comprising aminoacid residues 45 to 54 of SEQ ID NO:44, a heavy chain CDR2 sequencecomprising amino acid residues 69 to 85 of SEQ ID NO:44, and a heavychain CDR3 sequence comprising amino acid residues 118 to 125 of SEQ IDNO:44; 2) an antibody which comprises a light chain CDR1 sequencecomprising amino acid residues 44 to 54 of SEQ ID NO:47, a light chainCDR2 sequence comprising amino acid residues 70 to 76 of SEQ ID NO:47, alight chain CDR3 sequence comprising amino acid residues 109 to 117 ofSEQ ID NO:47, a heavy chain CDR1 sequence comprising amino acid residues45 to 54 of SEQ ID NO:48, a heavy chain CDR2 sequence comprising aminoacid residues 69 to 85 of SEQ ID NO:48, and a heavy chain CDR3 sequencecomprising amino acid residues 118 to 126 of SEQ ID NO:48; 3) anantibody which comprises a light chain CDR1 sequence comprising aminoacid residues 44 to 58 of SEQ ID NO:51, a light chain CDR2 sequencecomprising amino acid residues 74 to 80 of SEQ ID NO:51, a light chainCDR3 sequence comprising amino acid residues 113 to 121 of SEQ ID NO:51,a heavy chain CDR1 sequence comprising amino acid residues 45 to 54 ofSEQ ID NO:52, a heavy chain CDR2 sequence comprising amino acid residues69 to 85 of SEQ ID NO:52, and a heavy chain CDR3 sequence comprisingamino acid residues 118 to 130 of SEQ ID NO:52; and 4) an antibody whichcomprises a light chain CDR1 sequence comprising amino acid residues 44to 54 of SEQ ID NO:55, a light chain CDR2 sequence comprising amino acidresidues 70 to 76 of SEQ ID NO:55, a light chain CDR3 sequencecomprising amino acid residues 109 to 117 of SEQ ID NO:55, a heavy chainCDR1 sequence comprising amino acid residues 45 to 54 of SEQ ID NO:56, aheavy chain CDR2 sequence comprising amino acid residues 69 to 85 of SEQID NO:56, and a heavy chain CDR3 sequence comprising amino acid residues118 to 122 of SEQ ID NO:56;

Further details of the antibodies of the subject invention are providedin the section below entitled “Antibodies”.

The subject invention further provides an assay kit comprising theantibody, as well as an assay device comprising the antibody.Preferably, the assay device is a lateral flow assay device. Furtherdetails of the assay kits and assay devices are provided below in thesection entitled “Assay Kits and Devices”.

The invention further provides a method of detecting aripiprazole in asample. The method comprises: (i) contacting a sample with an antibodyaccording to the subject invention which is labeled with a detectablemarker, wherein the labeled antibody and aripiprazole present in thesample form a labeled complex; and (ii) detecting the labeled complex soas to detect aripiprazole in the sample. Further details of the methodof detecting aripiprazole in accordance with the subject invention areprovided in the section below entitled “Immunoassays”.

Further provided is a competitive immunoassay method for detectingaripiprazole in a sample. The method comprises: (i) contacting a samplewith an antibody according to the subject invention, and witharipiprazole or a competitive binding partner of aripiprazole, whereinone of the antibody and the aripiprazole or competitive binding partnerthereof is labeled with a detectable marker, and wherein samplearipiprazole competes with the aripiprazole or competitive bindingpartner thereof for binding to the antibody; and (ii) detecting thelabel so as to detect sample aripiprazole. Further details of thecompetitive immunoassay method of detecting aripiprazole in accordancewith the subject invention are provided in the section below entitled“Immunoassays”.

In a preferred embodiment of the subject invention, the detection ofaripiprazole is accompanied by the detection of one or more analytes inaddition to aripiprazole. Preferably the one or more analytes areanti-psychotic drugs other than aripiprazole, and more preferably theanti-psychotic drugs other than aripiprazole are selected from the groupconsisting of: risperidone, paliperidone, quetiapine, olanzapine, andmetabolites thereof.

As discussed above, the antibodies of the subject invention can be usedin assays to detect the presence and/or amount of the anti-psychoticdrug in patient samples. Such detection permits therapeutic drugmonitoring enabling all of the benefits thereof. Detection of levels ofanti-psychotic drugs may be useful for many purposes, each of whichrepresents another embodiment of the subject invention, including:determination of patient adherence or compliance with prescribedtherapy; use as a decision tool to determine whether a patient should beconverted from an oral anti-psychotic regimen to a long-actinginjectable anti-psychotic regimen; use as a decision tool to determineif the dose level or dosing interval of oral or injectableanti-psychotics should be increased or decreased to ensure attainment ormaintenance of efficacious or safe drug levels; use as an aid in theinitiation of anti-psychotic drug therapy by providing evidence of theattainment of minimum pK levels; use to determine bioequivalence ofanti-psychotic drug in multiple formulations or from multiple sources;use to assess the impact of polypharmacy and potential drug-druginteractions; and use as an indication that a patient should be excludedfrom or included in a clinical trial and as an aid in the subsequentmonitoring of adherence to clinical trial medication requirements.

Antibodies

The present invention provides an isolated antibody which binds toaripiprazole. The term “antibody” refers to a specific protein capableof binding an antigen or portion thereof (in accordance with thisinvention, capable of binding to an anti-psychotic drug or metabolitethereof). An antibody is produced in response to an immunogen which mayhave been introduced into a host, e.g., an animal or a human, byinjection. The generic term “antibody” includes polyclonal antibodies,monoclonal antibodies, and antibody fragments.

“Antibody” or “antigen-binding antibody fragment” refers to an intactantibody, or a fragment thereof, that competes with the intact antibodyfor binding. Generally speaking, an antibody or antigen-binding antibodyfragment, is said to specifically bind an antigen when the dissociationconstant is less than or equal to 1 μM, preferably less than or equal to100 nM and most preferably less than or equal to 10 nM. Binding can bemeasured by methods know to those skilled in the art, an example beingthe use of a BIAcore™ instrument.

Antibodies are made up of two heavy chains and two light chains. Eachheavy chain has one variable domain or region (V_(H)) followed by aconstant domain or region (C_(H)1), a hinge region, and two moreconstant domains or regions (C_(H)2 and C_(H)3). Each light chain hasone variable domain or region (V_(L)) and one constant domain or region(C_(L)). The variable domains or regions of the heavy and light chainsform the paratope of the antibody (a structure analogous to a lock),which is specific for a particular epitope (similarly analogous to akey), allowing the paratope and the epitope to bind together withprecision. Within the variable domain, variable loops of β-strands,three each on the light and heavy chains, are responsible for binding tothe antigen. These loops are referred to as the complementaritydetermining regions (CDRs, namely CDR1, CDR2, and CDR3).

Antibody fragments comprise a portion of an intact antibody, preferablythe antigen binding or variable region of the intact antibody. Bindingfragments include Fab, Fab′, F(ab′)₂, and Fv fragments; diabodies;minibodies; linear antibodies; single-chain antibody molecules (e.g.,scFV); and multispecific antibodies formed from antibody fragments. Anantibody other than a “bispecific” or “bifunctional” antibody isunderstood to have each of its binding sites identical.

As used herein, “epitope” includes any protein determinant capable ofspecific binding to an immunoglobulin or T-cell receptor. Epitopicdeterminants usually consist of chemically active surface groupings ofmolecules such as amino acids or sugar side chains and usually havespecific three dimensional structural characteristics, as well asspecific charge characteristics. Two antibodies are said to “bind thesame epitope” (“compete”) if one antibody is shown to compete with thesecond antibody in a competitive binding assay, by any of the methodswell known to those skilled in the art (such as the BIAcore™ methodreferred to above). In reference to a hapten (such as aripiprazole orother anti-psychotic drug), an antibody can be generated against thenon-antigenic hapten molecule by conjugating the hapten to animmunogenic carrier. An antibody is then generated which recognizes an“epitope” defined by the hapten.

“Isolated” when used in the context of an antibody means altered “by thehand of man” from any natural state; i.e., that, if it occurs in nature,it has been changed or removed from its original environment, or both.For example, a naturally occurring antibody naturally present in aliving animal in its natural state is not “isolated”, but the sameantibody separated from the coexisting materials of its natural state is“isolated”, as the term is employed herein. Antibodies may occur in acomposition, such as an immunoassay reagent, which are not naturallyoccurring compositions, and therein remain isolated antibodies withinthe meaning of that term as it is employed herein.

“Cross-reactivity” refers to the reaction of an antibody with an antigenthat was not used to induce that antibody.

Preferably, the antibody of the subject invention will bind to the drugand any desired pharmacologically active metabolites. By altering thelocation of the attachment of an immunogenic carrier in a drugconjugate, selectivity and cross-reactivity with metabolites can beengineered into the antibodies. For aripiprazole, cross-reactivity withdehydroaripiprazole may be desirable. Antibodies may be generated thatdetect both aripiprazole and dehydroaripiprazole, or antibodies may begenerated that detect each separately (thus defining the antibody“specific binding” properties). An antibody specifically binds one ormore compounds when its binding of the one or more compounds isequimolar or substantially equimolar.

The antibodies herein are described by the nucleotide and amino acidsequences of their variable domains. Each was generated by inoculating ahost with a conjugate comprising an anti-psychotic drug conjugated to animmunogenic carrier. Having now provided the nucleotide and amino acidsequences thereof, the antibodies can be produced by the recombinantmethods such as are described in U.S. Pat. No. 4,166,452.

Antibody fragments which contain specific binding sites for theanti-psychotic drug may also be generated. Such fragments include, butare not limited to, the F(ab′)₂ fragments which can be produced bypepsin digestion of the antibody molecule and the Fab fragments whichcan be generated by reducing the disulfide bridges of the F(ab′)₂fragments. Alternatively, Fab expression libraries may be constructed toallow rapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse et al., Science 256:1270-1281 (1989)). Fab, Fvand ScFv antibody fragments can all be expressed in and secreted fromEscherichia coli, allowing for the production of large amounts of thesefragments. Alternatively, Fab′-SH fragments can be directly recoveredfrom E. coli and chemically coupled to form F(ab′)₂ fragments (Carter etal., BioTechnology 10:163-167 (1992)). Other techniques for theproduction of antibody fragments are known to those skilled in the art.Single chain Fv fragments (scFv) are also envisioned (see U.S. Pat. Nos.5,761,894 and 5,587,458). Fv and sFv fragments are the only species withintact combining sites that are devoid of constant regions; thus, theyare likely to show reduced non-specific binding. The antibody fragmentmay also be a “linear antibody” e.g., as described in U.S. Pat. No.5,642,870, for example. Such linear antibody fragments may bemonospecific or bispecific.

Assay Kits and Devices

An assay kit (also referred to as a reagent kit) can also be providedcomprising an antibody as described above. A representative reagent kitmay comprise an antibody that binds to the anti-psychotic drug,aripiprazole, a complex comprising an analog of an anti-psychotic drugor a derivative thereof coupled to a labeling moiety, and may optionallyalso comprise one or more calibrators comprising a known amount of ananti-psychotic drug or a related standard.

The phrase “assay kit” refers to an assembly of materials and reagentsthat is used in performing an assay. The reagents can be provided inpackaged combination in the same or in separate containers, depending ontheir cross-reactivities and stabilities, and in liquid or inlyophilized form. The amounts and proportions of reagents provided inthe kit can be selected so as to provide optimum results for aparticular application. An assay kit embodying features of the presentinvention comprises antibodies which bind aripiprazole. The kit mayfurther comprise competitive binding partners of aripiprazole andcalibration and control materials.

The phrase “calibration and control material” refers to any standard orreference material containing a known amount of an analyte. A samplesuspected of containing an analyte and the corresponding calibrationmaterial are assayed under similar conditions. The concentration ofanalyte is calculated by comparing the results obtained for the unknownspecimen with the results obtained for the standard. This is commonlydone by constructing a calibration curve.

Antibodies embodying features of the present invention can be includedin a kit, container, pack, or dispenser together with instructions fortheir utilization. When the antibodies are supplied in a kit, thedifferent components of the immunoassay may be packaged in separatecontainers and admixed prior to use. Such packaging of the componentsseparately may permit long-term storage without substantiallydiminishing the functioning of the active components. Furthermore,reagents can be packaged under inert environments, e.g., under apositive pressure of nitrogen gas, argon gas, or the like, which isespecially preferred for reagents that are sensitive to air and/ormoisture.

Reagents included in kits embodying features of the present inventioncan be supplied in all manner of containers such that the activities ofthe different components are substantially preserved while thecomponents themselves are not substantially adsorbed or altered by thematerials of the container. Suitable containers include, but are notlimited to, ampules, bottles, test tubes, vials, flasks, syringes,envelopes, e.g., foil-lined, and the like. The containers may becomprised of any suitable material including, but not limited to, glass,organic polymers, e.g., polycarbonate, polystyrene, polyethylene, etc.,ceramic, metal, e.g., aluminum, metal alloys, e.g., steel, cork, and thelike. In addition, the containers may comprise one or more sterileaccess ports, e.g., for access via a needle, such as may be provided bya septum. Preferred materials for septa include rubber andpolytetrafluoroethylene of the type sold under the trade name TEFLON byDuPont (Wilmington, Del.). In addition, the containers may comprise twoor more compartments separated by partitions or membranes that can beremoved to allow mixing of the components.

Reagent kits embodying features of the present invention may also besupplied with instructional materials. Instructions may be printed,e.g., on paper and/or supplied in an electronically-readable medium.Alternatively, instructions may be provided by directing a user to aninternet website, e.g., specified by the manufacturer or distributor ofthe kit and/or via electronic mail.

The antibody may also be provided as part of an assay device. Such assaydevices include lateral flow assay devices. A common type of disposablelateral flow assay device includes a zone or area for receiving theliquid sample, a conjugate zone, and a reaction zone. These assaydevices are commonly known as lateral flow test strips. They employ aporous material, e.g., nitrocellulose, defining a path for fluid flowcapable of supporting capillary flow. Examples include those shown inU.S. Pat. Nos. 5,559,041, 5,714,389, 5,120,643, and 6,228,660 all ofwhich are incorporated herein by reference in their entireties.

Another type of assay device is a non-porous assay device havingprojections to induce capillary flow. Examples of such assay devicesinclude the open lateral flow device as disclosed in PCT InternationalPublication Nos. WO 2003/103835, WO 2005/089082, WO 2005/118139, and WO2006/137785, all of which are incorporated herein by reference in theirentireties.

In a non-porous assay device, the assay device generally has at leastone sample addition zone, at least one conjugate zone, at least onereaction zone, and at least one wicking zone. The zones form a flow pathby which sample flows from the sample addition zone to the wicking zone.Also included are capture elements, such as antibodies, in the reactionzone, capable of binding to the analyte, optionally deposited on thedevice (such as by coating); and a labeled conjugate material alsocapable of participating in reactions that will enable determination ofthe concentration of the analyte, deposited on the device in theconjugate zone, wherein the labeled conjugate material carries a labelfor detection in the reaction zone. The conjugate material is dissolvedas the sample flows through the conjugate zone forming a conjugate plumeof dissolved labeled conjugate material and sample that flows downstreamto the reaction zone. As the conjugate plume flows into the reactionzone, the conjugated material will be captured by the capture elementssuch as via a complex of conjugated material and analyte (as in a“sandwich” assay) or directly (as in a “competitive” assay). Unbounddissolved conjugate material will be swept past the reaction zone intothe at least one wicking zone. Such devices can include projections ormicropillars in the flow path.

An instrument such as that disclosed in US Patent Publication Nos.US20060289787A1 and US 20070231883A1, and U.S. Pat. Nos. 7,416,700 and6,139,800, all of which are incorporated herein by reference in theirentireties, is able to detect the bound conjugated material in thereaction zone. Common labels include fluorescent dyes that can bedetected by instruments which excite the fluorescent dyes andincorporate a detector capable of detecting the fluorescent dyes.

Immunoassays

The antibodies thus produced can be used in immunoassays torecognize/bind to the anti-psychotic drug, thereby detecting thepresence and/or amount of the drug in a patient sample. Preferably, theassay format is a competitive immunoassay format. Such an assay formatand other assays are described, among other places, in Hampton et al.(Serological Methods, A Laboratory Manual, APS Press, St. Paul, Minn.1990) and Maddox et al. (J. Exp. Med. 158:12111, 1983).

The term “analyte” refers to any substance or group of substances, thepresence or amount of which is to be determined. Representativeanti-psychotic drug analytes include, but are not limited to,risperidone, paliperidone, olanzapine, aripiprazole, and quetiapine.

The term “competitive binding partner” refers to a substance or group ofsubstances, such as may be employed in a competitive immunoassay, whichbehave similarly to an analyte with respect to binding affinity to anantibody. Representative competitive binding partners include, but arenot limited to, anti-psychotic drug derivatives and the like.

The term “detecting” when used with an analyte refers to anyquantitative, semi-quantitative, or qualitative method as well as to allother methods for determining an analyte in general, and ananti-psychotic drug in particular. For example, a method that merelydetects the presence or absence of an anti-psychotic drug in a samplelies within the scope of the present invention, as do methods thatprovide data as to the amount or concentration of the anti-psychoticdrug in the sample. The terms “detecting”, “determining”, “identifying”,and the like are used synonymously herein, and all lie within the scopeof the present invention.

A preferred embodiment of the subject invention is a competitiveimmunoassay wherein antibodies which bind the anti-psychotic drug, orthe drug or competitive binding partner thereof, are attached to a solidsupport (such as the reaction zone in a lateral flow assay device) andlabeled drug or competitive binding partner thereof, or labeledantibody, respectively, and a sample derived from the host are passedover the solid support and the amount of label detected attached to thesolid support can be correlated to a quantity of drug in the sample.

Any sample that is suspected of containing an analyte, e.g., ananti-psychotic drug, can be analyzed in accordance with the methods ofthe presently preferred embodiments. The sample can be pretreated ifdesired and can be prepared in any convenient medium that does notinterfere with the assay. Preferably, the sample comprises an aqueousmedium such as a body fluid from a host, most preferably plasma orserum.

It is to be understood that all manner of immunoassays employingantibodies are contemplated for use in accordance with the presentlypreferred embodiments, including assays in which antibodies are bound tosolid phases and assays in which antibodies are in liquid media. Methodsof immunoassays that can be used to detect analytes using antibodiesembodying features of the present invention include, but are not limitedto, competitive (reagent limited) assays wherein labeled analyte(analyte analog) and analyte in a sample compete for antibodies andsingle-site immunometric assays wherein the antibody is labeled; and thelike.

All examples were carried out using standard techniques, which are wellknown and routine to those of skill in the art, except where otherwisedescribed in detail. Routine molecular biology techniques of thefollowing examples can be carried out as described in standardlaboratory manuals, such as Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd Ed., Cold Spring Habor Laboratory Press, ColdSpring Harbor, N.Y. (1989).

Copending applications entitled “Haptens of Aripiprazole” (AttorneyDocket No. PRD3265USPSP/USNP, U.S. Provisional Patent Appl. No.61/691,450, filed Aug. 21, 2012, and US 20140163206, filed Aug. 20,2013), “Haptens of Olanzapine” (Attorney Docket No. PRD3266USPSP/USNP,U.S. Provisional Patent Appl. No. 61/691,454, filed Aug. 21, 2012, andUS 20140213766, filed Aug. 20, 2013), “Haptens of Paliperidone”(Attorney Docket No. PRD3267USPSP/USNP, U.S. Provisional Patent Appl.No. 61/691,459, filed Aug. 21, 2012, and US 20140213767, filed Aug. 20,2013), “Haptens of Quetiapine” (Attorney Docket No. PRD3268USPSP/USNP,U.S. Provisional Patent Appl. No. 61/691,462, filed Aug. 21, 2012, andUS 20140221616, filed Aug. 20, 2013), “Haptens of Risperidone andPaliperidone” (Attorney Docket No. PRD3269USPSP/USNP, U.S. ProvisionalPatent Appl. No. 61/691,469, filed Aug. 21, 2012, and US 20140155585,Aug. 20, 2013), “Antibodies to Aripiprazole Haptens and Use Thereof”(Attorney Docket No. CDS5128USPSP/USNP, U.S. Provisional Patent Appl.No. 61/691,544, filed Aug. 21, 2012, and US 20140057299, filed Aug. 20,2013), “Antibodies to Olanzapine Haptens and Use Thereof” (AttorneyDocket No. CDS5132USPSP/USNP, U.S. Provisional Patent Appl. No.61/691,572, filed Aug. 21, 2012, US 20140057303, filed Aug. 20, 2013),“Antibodies to Paliperidone Haptens and Use Thereof” (Attorney DocketNo. CDS5126USPSP/USNP, U.S. Provisional Patent Appl. No. 61/691,634,filed Aug. 21, 2012, and US 20140057297, filed Aug. 20, 2013),“Antibodies to Quetiapine Haptens and Use Thereof” (Attorney Docket No.CDS5134USPSP/USNP, U.S. Provisional Patent Appl. No. 61/691,598, filedAug. 21, 2012, and US 20140057305, filed Aug. 20, 2013), “Antibodies toRisperidone Haptens and Use Thereof” (Attorney Docket No.CDS5130USPSP/USNP, U.S. Provisional Patent Appl. No. 61/691,615, filedAug. 21, 2012, and US 20140057301, filed Aug. 20, 2013), “Antibodies toOlanzapine and Use Thereof” (Attorney Docket No. CDS5133USPSP/USNP, U.S.Provisional Patent Appl. No. 61/691,645, filed Aug. 21, 2012, and US20140057304, filed Aug. 20, 2013), “Antibodies to Paliperidone and UseThereof” (Attorney Docket No. CDS5127USPSP/USNP, U.S. Provisional PatentAppl. No. 61/691,692, filed Aug. 21, 2012, and US 20140057298, filedAug. 20, 2013), “Antibodies to Quetiapine and Use Thereof” (AttorneyDocket No. CDS5135USPSP/USNP, U.S. Provisional Patent Appl. No.61/691,659, filed Aug. 21, 2012, and US 20140057306, filed Aug. 20,2013), “Antibodies to Risperidone and Use Thereof” (Attorney Docket No.CDS5131USPSP/USNP, U.S. Provisional Patent Appl. No. 61/691,675, filedAug. 21, 2012, and US 20140057302, filed Aug. 20, 2013), and “Antibodiesto Risperidone and Use Thereof” (Attorney Docket No. CDS5145USPSP/USNP,U.S. Provisional Patent Appl. No. 61/790,880, filed Mar. 15, 2013, andUS 20140057302, filed Aug. 20, 2013) are all incorporated herein byreference in their entireties.

EXAMPLE 1 Antibodies to Aripiprazole

Antibody 17.3 Clone 3D7

The hybridoma designated 17.3 clone 3D7 secretes a monoclonal antibody(mAb) specific for aripiprazole. The antibody is designated 17.3 clone3D7. The nucleotide sequence of mAb 17.3 clone 3D7's light chainvariable region (V_(L)) is designated SEQ ID NO:41 and that of the heavychain variable region (V_(H)) is designated SEQ ID NO:42. Within mAb17.3 clone 3D7's V_(L), nucleotides 136-165 of SEQ ID NO:41 representthe first complementarity determining region (CDR1); nucleotides 211-231of SEQ ID NO:41 represent the second complementarity determining region(CDR2); and nucleotides 328-354 of SEQ ID NO:41 represent the thirdcomplementarity determining region (CDR3). Within mAb 17.3 clone 3D7'sV_(H), nucleotides 133-162 of SEQ ID NO:42 represent the firstcomplementarity determining region (CDR1); nucleotides 205-255 of SEQ IDNO:42 represent the second complementarity determining region (CDR2);and nucleotides 352-375 of SEQ ID NO:42 represent the thirdcomplementarity determining region (CDR3).

The corresponding predicted amino acid sequences of mAb 17.3 clone 3D7'svariable chain regions were also determined, and are designated SEQ IDNO:43 (light chain) and SEQ ID NO:44 (heavy chain). Within mAb 17.3clone 3D7's V_(L), amino acid residues 46-55 of SEQ ID NO:43 representthe first complementarity determining region (CDR1); amino acid residues71-77 of SEQ ID NO:43 represent the second complementarity determiningregion (CDR2); and amino acid residues 110-118 of SEQ ID NO:43 representthe third complementarity determining region (CDR3). Within mAb 17.3clone 3D7's V_(H), amino acid residues 45-54 of SEQ ID NO:44 representthe first complementarity determining region (CDR1); amino acid residues69-85 of SEQ ID NO:44 represent the second complementarity determiningregion (CDR2); and amino acid residues 118-125 of SEQ ID NO:44 representthe third complementarity determining region (CDR3).

Antibody 17.3 Clone 5C7 (First)

The hybridoma designated 17.3 clone 5C7 (first) secretes a monoclonalantibody (mAb) specific for aripiprazole. The antibody is designated17.3 clone 5C7 (first). The nucleotide sequence of mAb 17.3 clone 5C7(first)'s light chain variable region (V_(L)) is designated SEQ ID NO:45and that of the heavy chain variable region (V_(H)) is designated SEQ IDNO:46. Within mAb 17.3 clone 5C7 (first)'s V_(L), nucleotides 130-162 ofSEQ ID NO:45 represent the first complementarity determining region(CDR1); nucleotides 208-228 of SEQ ID NO:45 represent the secondcomplementarity determining region (CDR2); and nucleotides 325-351 ofSEQ ID NO:45 represent the third complementarity determining region(CDR3). Within mAb 17.3 clone 5C7 (first)'s V_(H), nucleotides 133-162of SEQ ID NO:46 represent the first complementarity determining region(CDR1); nucleotides 205-255 of SEQ ID NO:46 represent the secondcomplementarity determining region (CDR2); and nucleotides 352-378 ofSEQ ID NO:46 represent the third complementarity determining region(CDR3).

The corresponding predicted amino acid sequences of mAb 17.3 clone 5C7(first)'s variable chain regions were also determined, and aredesignated SEQ ID NO:47 (light chain) and SEQ ID NO:48 (heavy chain).Within mAb 17.3 clone 5C7 (first)'s V_(L), amino acid residues 44-54 ofSEQ ID NO:47 represent the first complementarity determining region(CDR1); amino acid residues 70-76 of SEQ ID NO:47 represent the secondcomplementarity determining region (CDR2); and amino acid residues109-117 of SEQ ID NO:47 represent the third complementarity determiningregion (CDR3). Within mAb 17.3 clone 5C7 (first)'s V_(H), amino acidresidues 45-54 of SEQ ID NO:48 represent the first complementaritydetermining region (CDR1); amino acid residues 69-85 of SEQ ID NO:48represent the second complementarity determining region (CDR2); andamino acid residues 118-126 of SEQ ID NO:48 represent the thirdcomplementarity determining region (CDR3).

Antibody 17.3 Clone 5C7 (Second)

The hybridoma designated 17.3 clone 5C7 (second) secretes a monoclonalantibody (mAb) specific for aripiprazole. The antibody is designated17.3 clone 5C7 (second). The nucleotide sequence of mAb 17.3 clone 5C7(second)'s light chain variable region (V_(L)) is designated SEQ IDNO:49 and that of the heavy chain variable region (V_(H)) is designatedSEQ ID NO:50. Within mAb 17.3 clone 5C7 (second)'s V_(L), nucleotides130-174 of SEQ ID NO:49 represent the first complementarity determiningregion (CDR1); nucleotides 220-240 of SEQ ID NO:49 represent the secondcomplementarity determining region (CDR2); and nucleotides 337-363 ofSEQ ID NO:49 represent the third complementarity determining region(CDR3). Within mAb 17.3 clone 5C7 (second)'s V_(H), nucleotides 133-162of SEQ ID NO:50 represent the first complementarity determining region(CDR1); nucleotides 205-255 of SEQ ID NO:50 represent the secondcomplementarity determining region (CDR2); and nucleotides 352-390 ofSEQ ID NO:50 represent the third complementarity determining region(CDR3).

The corresponding predicted amino acid sequences of mAb 17.3 clone 5C7(second)'s variable chain regions were also determined, and aredesignated SEQ ID NO:51 (light chain) and SEQ ID NO:52 (heavy chain).Within mAb 17.3 clone 5C7 (second)'s V_(L), amino acid residues 44-58 ofSEQ ID NO:51 represent the first complementarity determining region(CDR1); amino acid residues 74-80 of SEQ ID NO:51 represent the secondcomplementarity determining region (CDR2); and amino acid residues113-121 of SEQ ID NO:51 represent the third complementarity determiningregion (CDR3). Within mAb 17.3 clone 5C7 (second)'s V_(H), amino acidresidues 45-54 of SEQ ID NO:52 represent the first complementaritydetermining region (CDR1); amino acid residues 69-85 of SEQ ID NO:52represent the second complementarity determining region (CDR2); andamino acid residues 118-130 of SEQ ID NO:52 represent the thirdcomplementarity determining region (CDR3).

Antibody 17.3 Clone 5C7 (Third)

The hybridoma designated 17.3 clone 5C7 (third) secretes a monoclonalantibody (mAb) specific for aripiprazole. The antibody is designated17.3 clone 5C7 (third). The nucleotide sequence of mAb 17.3 clone 5C7(third)'s light chain variable region (V_(L)) is designated SEQ ID NO:53and that of the heavy chain variable region (V_(H)) is designated SEQ IDNO:54. Within mAb 17.3 clone 5C7 (third)'s V_(L), nucleotides 130-162 ofSEQ ID NO:53 represent the first complementarity determining region(CDR1); nucleotides 208-228 of SEQ ID NO:53 represent the secondcomplementarity determining region (CDR2); and nucleotides 325-351 ofSEQ ID NO:53 represent the third complementarity determining region(CDR3). Within mAb 17.3 clone 5C7 (third)'s V_(H), nucleotides 133-162of SEQ ID NO:54 represent the first complementarity determining region(CDR1); nucleotides 205-255 of SEQ ID NO:54 represent the secondcomplementarity determining region (CDR2); and nucleotides 352-366 ofSEQ ID NO:54 represent the third complementarity determining region(CDR3).

The corresponding predicted amino acid sequences of mAb 17.3 clone 5C7(third)'s variable chain regions were also determined, and aredesignated SEQ ID NO:55 (light chain) and SEQ ID NO:56 (heavy chain).Within mAb 17.3 clone 5C7 (third)'s V_(L), amino acid residues 44-54 ofSEQ ID NO:55 represent the first complementarity determining region(CDR1); amino acid residues 70-76 of SEQ ID NO:55 represent the secondcomplementarity determining region (CDR2); and amino acid residues109-117 of SEQ ID NO:55 represent the third complementarity determiningregion (CDR3). Within mAb 17.3 clone 5C7 (third)'s V_(H), amino acidresidues 45-54 of SEQ ID NO:56 represent the first complementaritydetermining region (CDR1); amino acid residues 69-85 of SEQ ID NO:56represent the second complementarity determining region (CDR2); andamino acid residues 118-122 of SEQ ID NO:56 represent the thirdcomplementarity determining region (CDR3).

EXAMPLE 2 Antibodies to Olanzapine

Antibody 11.1 Clone 35

The hybridoma designated 11.1 clone 35 secretes a monoclonal antibody(mAb) specific for olanzapine. The antibody is designated 11.1 clone 35.The nucleotide sequence of mAb 11.1 clone 35's light chain variableregion (V_(L)) is designated SEQ ID NO:9 and that of the heavy chainvariable region (V_(H)) is designated SEQ ID NO:10. Within mAb 11.1clone 35's V_(L), nucleotides 130-162 of SEQ ID NO:9 represent the firstcomplementarity determining region (CDR1); nucleotides 208-228 of SEQ IDNO:9 represent the second complementarity determining region (CDR2); andnucleotides 325-351 of SEQ ID NO:9 represent the third complementaritydetermining region (CDR3). Within mAb 11.1 clone 35's V_(H), nucleotides133-162 of SEQ ID NO:10 represent the first complementarity determiningregion (CDR1); nucleotides 205-255 of SEQ ID NO:10 represent the secondcomplementarity determining region (CDR2); and nucleotides 352-366 ofSEQ ID NO:10 represent the third complementarity determining region(CDR3).

The corresponding predicted amino acid sequences of mAb 11.1 clone 35'svariable chain regions were also determined, and are designated SEQ IDNO:11 (light chain) and SEQ ID NO:12 (heavy chain). Within mAb 11.1clone 35's V_(L), amino acid residues 44-54 of SEQ ID NO:11 representthe first complementarity determining region (CDR1); amino acid residues70-76 of SEQ ID NO:11 represent the second complementarity determiningregion (CDR2); and amino acid residues 109-117 of SEQ ID NO:11 representthe third complementarity determining region (CDR3). Within mAb 11.1clone 35's V_(H), amino acid residues 45-54 of SEQ ID NO:12 representthe first complementarity determining region (CDR1); amino acid residues69-85 of SEQ ID NO:12 represent the second complementarity determiningregion (CDR2); and amino acid residues 118-122 of SEQ ID NO:12 representthe third complementarity determining region (CDR3).

Antibody 11.1 Clone 61

The hybridoma designated 11.1 clone 61 secretes a monoclonal antibody(mAb) specific for olanzapine. The antibody is designated 11.1 clone 61.The nucleotide sequence of mAb 11.1 clone 61's light chain variableregion (V_(L)) is designated SEQ ID NO:13 and that of the heavy chainvariable region (V_(H)) is designated SEQ ID NO:14. Within mAb 11.1clone 61's V_(L), nucleotides 130-162 of SEQ ID NO:13 represent thefirst complementarity determining region (CDR1); nucleotides 208-228 ofSEQ ID NO:13 represent the second complementarity determining region(CDR2); and nucleotides 325-351 of SEQ ID NO:13 represent the thirdcomplementarity determining region (CDR3). Within mAb 11.1 clone 61'sV_(H), nucleotides 133-162 of SEQ ID NO:14 represent the firstcomplementarity determining region (CDR1); nucleotides 205-255 of SEQ IDNO:14 represent the second complementarity determining region (CDR2);and nucleotides 352-366 of SEQ ID NO:14 represent the thirdcomplementarity determining region (CDR3).

The corresponding predicted amino acid sequences of mAb 11.1 clone 61'svariable chain regions were also determined, and are designated SEQ IDNO:15 (light chain) and SEQ ID NO:16 (heavy chain). Within mAb 11.1clone 61's V_(L), amino acid residues 44-54 of SEQ ID NO:15 representthe first complementarity determining region (CDR1); amino acid residues70-76 of SEQ ID NO:15 represent the second complementarity determiningregion (CDR2); and amino acid residues 109-117 of SEQ ID NO:15 representthe third complementarity determining region (CDR3). Within mAb 11.1clone 61's V_(H), amino acid residues 45-54 of SEQ ID NO:16 representthe first complementarity determining region (CDR1); amino acid residues69-85 of SEQ ID NO:16 represent the second complementarity determiningregion (CDR2); and amino acid residues 118-122 of SEQ ID NO:16 representthe third complementarity determining region (CDR3).

Antibody 15.5 Clone 3F11 (First)

The hybridoma designated 15.5 clone 3F11 (first) secretes a monoclonalantibody (mAb) specific for olanzapine. The antibody is designated 15.5clone 3F11 (first). The nucleotide sequence of mAb 15.5 clone 3F11(first)'s light chain variable region (V_(L)) is designated SEQ ID NO:29and that of the heavy chain variable region (V_(H)) is designated SEQ IDNO:30. Within mAb 15.5 clone 3F11 (first)'s V_(L), nucleotides 130-162of SEQ ID NO:29 represent the first complementarity determining region(CDR1); nucleotides 208-228 of SEQ ID NO:29 represent the secondcomplementarity determining region (CDR2); and nucleotides 325-351 ofSEQ ID NO:29 represent the third complementarity determining region(CDR3). Within mAb 15.5 clone 3F11 (first)'s V_(H), nucleotides 130-162of SEQ ID NO:30 represent the first complementarity determining region(CDR1); nucleotides 205-252 of SEQ ID NO:30 represent the secondcomplementarity determining region (CDR2); and nucleotides 355-381 ofSEQ ID NO:30 represent the third complementarity determining region(CDR3).

The corresponding predicted amino acid sequences of mAb 15.5 clone 3F11(first)'s variable chain regions were also determined, and aredesignated SEQ ID NO:31 (light chain) and SEQ ID NO:32 (heavy chain).Within mAb 15.5 clone 3F11 (first)'s V_(L), amino acid residues 44-54 ofSEQ ID NO:31 represent the first complementarity determining region(CDR1); amino acid residues 70-76 of SEQ ID NO:31 represent the secondcomplementarity determining region (CDR2); and amino acid residues109-117 of SEQ ID NO:31 represent the third complementarity determiningregion (CDR3). Within mAb 15.5 clone 3F11 (first)'s V_(H), amino acidresidues 44-54 of SEQ ID NO:32 represent the first complementaritydetermining region (CDR1); amino acid residues 69-84 of SEQ ID NO:32represent the second complementarity determining region (CDR2); andamino acid residues 119-127 of SEQ ID NO:32 represent the thirdcomplementarity determining region (CDR3).

Antibody 15.5 Clone 3F11 (Second)

The hybridoma designated 15.5 clone 3F11 (second) secretes a monoclonalantibody (mAb) specific for olanzapine. The antibody is designated 15.5clone 3F11 (second). The nucleotide sequence of mAb 15.5 clone 3F11(second)'s light chain variable region (V_(L)) is designated SEQ IDNO:33 and that of the heavy chain variable region (V_(H)) is designatedSEQ ID NO:34. Within mAb 15.5 clone 3F11 (second)'s V_(L), nucleotides130-162 of SEQ ID NO:33 represent the first complementarity determiningregion (CDR1); nucleotides 208-228 of SEQ ID NO:33 represent the secondcomplementarity determining region (CDR2); and nucleotides 325-351 ofSEQ ID NO:33 represent the third complementarity determining region(CDR3). Within mAb 15.5 clone 3F11 (second)'s V_(H), nucleotides 133-162of SEQ ID NO:34 represent the first complementarity determining region(CDR1); nucleotides 205-261 of SEQ ID NO:34 represent the secondcomplementarity determining region (CDR2); and nucleotides 358-381 ofSEQ ID NO:34 represent the third complementarity determining region(CDR3).

The corresponding predicted amino acid sequences of mAb 15.5 clone 3F11(second)'s variable chain regions were also determined, and aredesignated SEQ ID NO:35 (light chain) and SEQ ID NO:36 (heavy chain).Within mAb 15.5 clone 3F11 (second)'s V_(L), amino acid residues 44-54of SEQ ID NO:35 represent the first complementarity determining region(CDR1); amino acid residues 70-76 of SEQ ID NO:35 represent the secondcomplementarity determining region (CDR2); and amino acid residues109-117 of SEQ ID NO:35 represent the third complementarity determiningregion (CDR3). Within mAb 15.5 clone 3F11 (second)'s V_(H), amino acidresidues 45-54 of SEQ ID NO:36 represent the first complementaritydetermining region (CDR1); amino acid residues 69-87 of SEQ ID NO:36represent the second complementarity determining region (CDR2); andamino acid residues 120-127 of SEQ ID NO:36 represent the thirdcomplementarity determining region (CDR3).

Antibody 15.5 Sub-Clone 4G9-1

The hybridoma designated 15.5 sub-clone 4G9-1 secretes a monoclonalantibody (mAb) specific for olanzapine. The antibody is designated 15.5sub-clone 4G9-1. The nucleotide sequence of mAb 15.5 sub-clone 4G9-1'slight chain variable region (V_(L)) is designated SEQ ID NO:37 and thatof the heavy chain variable region (V_(H)) is designated SEQ ID NO:38.Within mAb 15.5 sub-clone 4G9-1's V_(L), nucleotides 130-162 of SEQ IDNO:37 represent the first complementarity determining region (CDR1);nucleotides 208-228 of SEQ ID NO:37 represent the second complementaritydetermining region (CDR2); and nucleotides 325-351 of SEQ ID NO:37represent the third complementarity determining region (CDR3). WithinmAb 15.5 sub-clone 4G9-1's V_(H), nucleotides 130-162 of SEQ ID NO:38represent the first complementarity determining region (CDR1);nucleotides 205-252 of SEQ ID NO:38 represent the second complementaritydetermining region (CDR2); and nucleotides 358-381 of SEQ ID NO:38represent the third complementarity determining region (CDR3).

The corresponding predicted amino acid sequences of mAb 15.5 sub-clone4G9-1's variable chain regions were also determined, and are designatedSEQ ID NO:39 (light chain) and SEQ ID NO:40 (heavy chain). Within mAb15.5 sub-clone 4G9-1's V_(L), amino acid residues 44-54 of SEQ ID NO:39represent the first complementarity determining region (CDR1); aminoacid residues 70-76 of SEQ ID NO:39 represent the second complementaritydetermining region (CDR2); and amino acid residues 109-117 of SEQ IDNO:39 represent the third complementarity determining region (CDR3).Within mAb 15.5 sub-clone 4G9-1's V_(H), amino acid residues 44-54 ofSEQ ID NO:40 represent the first complementarity determining region(CDR1); amino acid residues 69-84 of SEQ ID NO:40 represent the secondcomplementarity determining region (CDR2); and amino acid residues120-127 of SEQ ID NO:40 represent the third complementarity determiningregion (CDR3).

EXAMPLE 3 Antibodies to Quetiapine

Antibody 13.2 Sub-Clone 89-3 (First)

The hybridoma designated 13.2 sub-clone 89-3 (first) secretes amonoclonal antibody (mAb) specific for quetiapine. The antibody isdesignated 13.2 sub-clone 89-3 (first). The nucleotide sequence of mAb13.2 sub-clone 89-3 (first)'s light chain variable region (V_(L)) isdesignated SEQ ID NO:17 and that of the heavy chain variable region(V_(H)) is designated SEQ ID NO:18. Within mAb 13.2 sub-clone 89-3(first)'s V_(L), nucleotides 127-174 of SEQ ID NO:17 represent the firstcomplementarity determining region (CDR1); nucleotides 220-240 of SEQ IDNO:17 represent the second complementarity determining region (CDR2);and nucleotides 337-363 of SEQ ID NO:17 represent the thirdcomplementarity determining region (CDR3). Within mAb 13.2 sub-clone89-3 (first)'s V_(H), nucleotides 133-162 of SEQ ID NO:18 represent thefirst complementarity determining region (CDR1); nucleotides 205-255 ofSEQ ID NO:18 represent the second complementarity determining region(CDR2); and nucleotides 352-387 of SEQ ID NO:18 represent the thirdcomplementarity determining region (CDR3).

The corresponding predicted amino acid sequences of mAb 13.2 sub-clone89-3 (first)'s variable chain regions were also determined, and aredesignated SEQ ID NO:19 (light chain) and SEQ ID NO:20 (heavy chain).Within mAb 13.2 sub-clone 89-3 (first)'s V_(L), amino acid residues43-58 of SEQ ID NO:19 represent the first complementarity determiningregion (CDR1); amino acid residues 74-80 of SEQ ID NO:19 represent thesecond complementarity determining region (CDR2); and amino acidresidues 113-121 of SEQ ID NO:19 represent the third complementaritydetermining region (CDR3). Within mAb 13.2 sub-clone 89-3 (first)'sV_(H), amino acid residues 45-54 of SEQ ID NO:20 represent the firstcomplementarity determining region (CDR1); amino acid residues 69-85 ofSEQ ID NO:20 represent the second complementarity determining region(CDR2); and amino acid residues 118-129 of SEQ ID NO:20 represent thethird complementarity determining region (CDR3).

Antibody 13.2 Sub-Clone 89-3 (Second)

The hybridoma designated 13.2 sub-clone 89-3 (second) secretes amonoclonal antibody (mAb) specific for quetiapine. The antibody isdesignated 13.2 sub-clone 89-3 (second). The nucleotide sequence of mAb13.2 sub-clone 89-3 (second)'s light chain variable region (V_(L)) isdesignated SEQ ID NO:21 and that of the heavy chain variable region(V_(H)) is designated SEQ ID NO:22. Within mAb 13.2 sub-clone 89-3(second)'s V_(L), nucleotides 127-174 of SEQ ID NO:21 represent thefirst complementarity determining region (CDR1); nucleotides 220-240 ofSEQ ID NO:21 represent the second complementarity determining region(CDR2); and nucleotides 337-363 of SEQ ID NO:21 represent the thirdcomplementarity determining region (CDR3). Within mAb 13.2 sub-clone89-3 (second)'s V_(H), nucleotides 133-162 of SEQ ID NO:22 represent thefirst complementarity determining region (CDR1); nucleotides 205-255 ofSEQ ID NO:22 represent the second complementarity determining region(CDR2); and nucleotides 367-387 of SEQ ID NO:22 represent the thirdcomplementarity determining region (CDR3).

The corresponding predicted amino acid sequences of mAb 13.2 sub-clone89-3 (second)'s variable chain regions were also determined, and aredesignated SEQ ID NO:23 (light chain) and SEQ ID NO:24 (heavy chain).Within mAb 13.2 sub-clone 89-3 (second)'s V_(L), amino acid residues43-58 of SEQ ID NO:23 represent the first complementarity determiningregion (CDR1); amino acid residues 74-80 of SEQ ID NO:23 represent thesecond complementarity determining region (CDR2); and amino acidresidues 113-121 of SEQ ID NO:23 represent the third complementaritydetermining region (CDR3). Within mAb 13.2 sub-clone 89-3 (second)'sV_(H), amino acid residues 45-54 of SEQ ID NO:24 represent the firstcomplementarity determining region (CDR1); amino acid residues 69-85 ofSEQ ID NO:24 represent the second complementarity determining region(CDR2); and amino acid residues 123-129 of SEQ ID NO:24 represent thethird complementarity determining region (CDR3).

Antibody 13.2 Sub-Clone 89-5

The hybridoma designated 13.2 sub-clone 89-5 secretes a monoclonalantibody (mAb) specific for quetiapine. The antibody is designated 13.2sub-clone 89-5. The nucleotide sequence of mAb 13.2 sub-clone 89-5'slight chain variable region (V_(L)) is designated SEQ ID NO:25 and thatof the heavy chain variable region (V_(H)) is designated SEQ ID NO:26.Within mAb 13.2 sub-clone 89-5's V_(L), nucleotides 127-174 of SEQ IDNO:25 represent the first complementarity determining region (CDR1);nucleotides 220-240 of SEQ ID NO:25 represent the second complementaritydetermining region (CDR2); and nucleotides 337-363 of SEQ ID NO:25represent the third complementarity determining region (CDR3). WithinmAb 13.2 sub-clone 89-5's V_(H), nucleotides 133-162 of SEQ ID NO:26represent the first complementarity determining region (CDR1);nucleotides 205-255 of SEQ ID NO:26 represent the second complementaritydetermining region (CDR2); and nucleotides 367-387 of SEQ ID NO:26represent the third complementarity determining region (CDR3).

The corresponding predicted amino acid sequences of mAb 13.2 sub-clone89-5's variable chain regions were also determined, and are designatedSEQ ID NO:27 (light chain) and SEQ ID NO:28 (heavy chain). Within mAb13.2 sub-clone 89-5's V_(L), amino acid residues 43-58 of SEQ ID NO:27represent the first complementarity determining region (CDR1); aminoacid residues 74-80 of SEQ ID NO:27 represent the second complementaritydetermining region (CDR2); and amino acid residues 113-121 of SEQ IDNO:27 represent the third complementarity determining region (CDR3).Within mAb 13.2 sub-clone 89-5's V_(H), amino acid residues 45-54 of SEQID NO:28 represent the first complementarity determining region (CDR1);amino acid residues 69-85 of SEQ ID NO:28 represent the secondcomplementarity determining region (CDR2); and amino acid residues123-129 of SEQ ID NO:28 represent the third complementarity determiningregion (CDR3).

EXAMPLE 4 Antibodies to Risperidone/Paliperidone

Antibody 5_9

The hybridoma designated 5_9 secretes a monoclonal antibody (mAb)specific for risperidone (and its metabolite paliperidone). The antibodyis designated 5-9. The nucleotide sequence of mAb 5-9's light chainvariable region (V_(L)) is designated SEQ ID NO:1 and that of the heavychain variable region (V_(H)) is designated SEQ ID NO:2. Within mAb5-9's V_(L), nucleotides 130-180 of SEQ ID NO:1 represent the firstcomplementarity determining region (CDR1); nucleotides 226-246 of SEQ IDNO:1 represent the second complementarity determining region (CDR2); andnucleotides 343-369 of SEQ ID NO:1 represent the third complementaritydetermining region (CDR3). Within mAb 5-9's V_(H), nucleotides 133-162of SEQ ID NO:2 represent the first complementarity determining region(CDR1); nucleotides 205-255 of SEQ ID NO:2 represent the secondcomplementarity determining region (CDR2); and nucleotides 352-366 ofSEQ ID NO:2 represent the third complementarity determining region(CDR3).

The corresponding predicted amino acid sequences of mAb 5-9's variablechain regions were also determined, and are designated SEQ ID NO:3(light chain) and SEQ ID NO:4 (heavy chain). Within mAb 5-9's V_(L),amino acid residues 44-60 of SEQ ID NO:3 represent the firstcomplementarity determining region (CDR1); amino acid residues 76-82 ofSEQ ID NO:3 represent the second complementarity determining region(CDR2); and amino acid residues 115-123 of SEQ ID NO:3 represent thethird complementarity determining region (CDR3). Within mAb 5-9's V_(H),amino acid residues 45-54 of SEQ ID NO:4 represent the firstcomplementarity determining region (CDR1); amino acid residues 69-85 ofSEQ ID NO:4 represent the second complementarity determining region(CDR2); and amino acid residues 118-122 of SEQ ID NO:4 represent thethird complementarity determining region (CDR3).

Antibody 5_5

The hybridoma designated 5_5 secretes a monoclonal antibody (mAb)specific for risperidone (and its metabolite paliperidone). The antibodyis designated 5-5. The nucleotide sequence of mAb 5-5's light chainvariable region (V_(L)) is designated SEQ ID NO:5 and that of the heavychain variable region (V_(H)) is designated SEQ ID NO:6. Within mAb5-5's V_(L), nucleotides 130-180 of SEQ ID NO:5 represent the firstcomplementarity determining region (CDR1); nucleotides 226-246 of SEQ IDNO:5 represent the second complementarity determining region (CDR2); andnucleotides 343-369 of SEQ ID NO:5 represent the third complementaritydetermining region (CDR3). Within mAb 5-9's V_(H), nucleotides 133-162of SEQ ID NO:6 represent the first complementarity determining region(CDR1); nucleotides 205-255 of SEQ ID NO:6 represent the secondcomplementarity determining region (CDR2); and nucleotides 352-366 ofSEQ ID NO:6 represent the third complementarity determining region(CDR3).

The corresponding predicted amino acid sequences of mAb 5-5's variablechain regions were also determined, and are designated SEQ ID NO:7(light chain) and SEQ ID NO:8 (heavy chain). Within mAb 5-5's V_(L),amino acid residues 44-60 of SEQ ID NO:7 represent the firstcomplementarity determining region (CDR1); amino acid residues 76-82 ofSEQ ID NO:7 represent the second complementarity determining region(CDR2); and amino acid residues 115-123 of SEQ ID NO:7 represent thethird complementarity determining region (CDR3). Within mAb 5-5's V_(H),amino acid residues 45-54 of SEQ ID NO:8 represent the firstcomplementarity determining region (CDR1); amino acid residues 69-85 ofSEQ ID NO:8 represent the second complementarity determining region(CDR2); and amino acid residues 118-122 of SEQ ID NO:8 represent thethird complementarity determining region (CDR3).

EXAMPLE 5 Competitive Immunoassays for Aripiprazole and MultiplexCompetitive Immunoassay for Aripiprazole, Olanzapine, Quetiapine, andRisperidone/Paliperidone

Following a series of immunizations with an aripiprazole immunogen,mouse tail bleeds were tested for reactivity using an ELISA. Hybridomasupernatants were also tested, and the ELISA data shown in Table 1 belowshows reactivity of several hybridomas (fusion partner was NSO cells).

TABLE 1 Plate 1 Dilution 1 2 3 4 5 400 3C1 3D7 5C6 5C7 5H11 400 12001200 3600 3600 10800 10800 400 0.8165 0.7299 0.196 3.2953 0.0373 4000.7057 0.5671 0.1525 2.9591 0.0371 1200 0.2413 0.2186 0.0701 1.92420.0348 1200 0.2474 0.2278 0.0653 1.7829 0.0336 3600 0.102 0.0963 0.04720.739 0.0288 3600 0.099 0.0954 0.051 0.7225 0.0281 10800 0.0534 0.05260.0381 0.2878 0.0215 10800 0.0644 0.0588 0.0411 0.2799 0.0326

Supernatant was then tested by competition ELISA to determine if thesignal was specific to either aripiprazole or dehydroaripiprazole. FIGS.1 and 2 show the results from two representative hybridomas, 3C1 and3D7. Data shows reactivity to both aripiprazole and dehydroaripiprazole.

FIG. 3 shows the competitive immunoassay format used on a lateral flowassay device in which the capture antibody, aripiprazole clone 5C7, wasdeposited on a chip along with a detection conjugate consisting ofaripiprazole conjugated to a fluorophore. In this competitive format asshow in FIG. 3, a low level of analyte (aripiprazole) results in highsignal, whereas a high level of analyte (aripiprazole) results in lowsignal.

FIG. 4 shows the chip design of a lateral flow assay device according toone embodiment of the subject invention. The device includes a zone orarea for receiving the sample, a conjugate zone (which contains desiredlabeled competitive binding partner(s)), and a reaction zone (eightareas within the reaction zone are indicated; each area can contain aseparate desired antibody). Sample flows from the sample zone throughthe conjugate zone and to the reaction zone.

FIGS. 5-8 show typical dose response curves for an aripiprazole positivecontrol (sample containing aripiprazole) generated with antibody 5C7deposited in reaction zone 2 and a labeled aripiprazole competitivebinding partner in the conjugate zone (FIG. 5), an olanzapine positivecontrol (sample containing olanzapine) generated with antibody 4G9-1deposited in reaction zone 4 and a labeled olanzapine competitivebinding partner in the conjugate zone (FIG. 6), a quetiapine positivecontrol (sample containing quetiapine) generated with antibody 11deposited in reaction zone 6 and a labeled quetiapine competitivebinding partner in the conjugate zone (FIG. 7), and a risperidonepositive control (sample containing risperidone) generated with antibody5-9 deposited in reaction zone 8 and a labeled risperidone competitivebinding partner in the conjugate zone (FIG. 8). The labeled competitivebinding partners in the conjugate zone compete with the drugs present inthe samples for binding to the antibodies. The amount of label isdetected and is an indication of the amount of drug present in thesample (the amount of signal being inversely proportional to the amountof drug in the sample—see FIG. 3).

In order to confirm that conjugates of labeled competitive bindingpartners do not bind to antibodies deposited in the reaction zones,negative controls were conducted by using samples containing no drugs.Referring to Table 2, a sample containing no aripiprazole is depositedin the sample zone and moves by capillary action through the conjugatezone (this time containing labeled olanzapine, labeled quetiapine, andlabeled risperidone, but no labeled aripiprazole) and to the reactionzone. The reaction zone again contains aripiprazole antibody (5C7) inreaction zone 2. Table 2 below shows the results, confirming that thereis no dose response and the olanzapine, quetiapine, and risperidoneconjugates that move by capillary action through the reaction zone donot bind to the aripiprazole antibody.

TABLE 2 Aripiprazole-Clone 5C7-Math Model 1 (0 ng/mL Conc.) Read PeakPeak Mean Assay- Reaction Posi- Mean Mean Back- MM Conj Zone tion AreaHeight ground ARIP- OLAN, QUET, ARIP 2 0.77 1.56 3.99 MM1 RISP ARIP-OLAN, QUET, 4 −0.02 0.06 4.14 MM1 RISP ARIP- OLAN, QUET, 6 0.09 0.104.29 MM1 RISP ARIP- OLAN, QUET, 8 0.13 0.12 4.61 MM1 RISP OtherConjugates do not bind to Aripiprazole

Referring to Table 3, a sample containing no olanzapine is deposited inthe sample zone and moves by capillary action through the conjugate zone(this time containing labeled aripiprazole, labeled quetiapine, andlabeled risperidone, but no labeled olanzapine) and to the reactionzone. The reaction zone again contains olanzapine antibody (4G9-1) inreaction zone 4. Table 3 below shows the results, confirming that thereis no dose response and the aripiprazole, quetiapine, and risperidoneconjugates that move by capillary action through the reaction zone donot bind to the olanzapine antibody.

TABLE 3 OLAN-Clone 4G9-1-Math Model 1 (0 ng/mL Conc.) Read Peak PeakMean Assay- Reaction Posi- Mean Mean Back- MM Conj Zone tion Area Heightground OLAN- ARIP, QUET, 2 −0.03 0.05 4.38 MM1 RISP OLAN- ARIP, QUET,OLAN 4 0.74 1.10 4.56 MM1 RISP OLAN- ARIP, QUET, 6 0.06 0.09 4.79 MM1RISP OLAN- ARIP, QUET, 8 0.11 0.13 5.17 MM1 RISP Other Conjugates do notbind to Olanzapine

Referring to Table 4, a sample containing no quetiapine is deposited inthe sample zone and moves by capillary action through the conjugate zone(this time containing labeled aripiprazole, labeled olanzapine, andlabeled risperidone, but no labeled quetiapine) and to the reactionzone. The reaction zone again contains quetiapine antibody (11) inreaction zone 6. Table 4 below shows the results, confirming that thereis no dose response and the aripiprazole, olanzapine, and risperidoneconjugates that move by capillary action through the reaction zone donot bind to the quetiapine antibody.

TABLE 4 Quetiapine-Clone 11-Math Model 1 (0 ng/mL Conc.) Read Peak PeakMean Assay- Reaction Posi- Mean Mean Back- MM Conj Zone tion Area Heightground QUET- ARIP, OLAN, 2 −0.01 0.07 3.85 MM1 RISP QUET- ARIP, OLAN, 40.01 0.12 4.01 MM1 RISP QUET- ARIP, OLAN, QUET 6 0.03 0.08 4.24 MM1 RISPQUET- ARIP, OLAN, 8 0.04 0.07 4.56 MM1 RISP Other Conjugates do not bindto Quetiapine

Referring to Table 5, a sample containing no risperidone is deposited inthe sample zone and moves by capillary action through the conjugate zone(this time containing labeled aripiprazole, labeled olanzapine, andlabeled quetiapine, but no labeled risperidone) and to the reactionzone. The reaction zone again contains risperidone antibody (5-9) inreaction zone 8. Table 5 below shows the results, confirming that thereis no dose response and the aripiprazole, olanzapine, and quetiapineconjugates that move by capillary action through the reaction zone donot bind to the risperidone antibody.

TABLE 5 Risperidone-Clone 5-9-Math Model 1 (0 ng/mL Conc.) Read PeakPeak Mean Assay- Reaction Posi- Mean Mean Back- MM Conj Zone tion AreaHeight ground RISP- ARIP, OLAN, 2 0.02 0.11 7.43 MM1 QUET RISP- ARIP,OLAN, 4 0.05 0.14 7.73 MM1 QUET RISP- ARIP, OLAN, 6 0.20 0.19 8.11 MM1QUET RISP- ARIP, OLAN, RISP 8 1.97 3.23 8.85 MM1 QUET Other Conjugatesdo not bind to Risperidone

In order to confirm that conjugates of labeled competitive bindingpartners bind only to their respective antibodies deposited in thereaction zones, additional negative controls were conducted by againusing samples containing no drugs. Referring to Table 6, a samplecontaining no aripiprazole is deposited in the sample zone and moves bycapillary action through the conjugate zone (this time containinglabeled aripiprazole) and to the reaction zone. The reaction zone againcontains aripiprazole antibody (5C7) in reaction zone 2, as well asolanzapine antibody (4G9-1) in reaction zone 4, quetiapine antibody (11)in reaction zone 6, and risperidone antibody (5-9) in reaction zone 8.Table 6 below shows the results, confirming that there is no doseresponse except to the aripiprazole antibody 5C7 (in reaction zone 2).

TABLE 6 Aripiprazole-Clone 5C7-Math Model 1 (0 ng/mL Conc.) Read PeakPeak Mean Assay- Reaction Posi- Mean Mean Back- MM Conj Zone tion AreaHeight ground ARIP- ARIP, OLAN, ARIP 2 60.34 97.53 5.44 MM1 QUET, RISPARIP- ARIP, OLAN, 4 2.86 3.91 11.66 MM1 QUET, RISP ARIP- ARIP, OLAN, 61.12 1.23 11.03 MM1 QUET, RISP ARIP- ARIP, OLAN, 8 3.14 4.19 12.94 MM1QUET, RISP Only the Aripiprazole Reaction Zone is binding

Referring to Table 7, a sample containing no olanzapine is deposited inthe sample zone and moves by capillary action through the conjugate zone(this time containing labeled olanzapine) and to the reaction zone. Thereaction zone again contains aripiprazole antibody (5C7) in reactionzone 2, as well as olanzapine antibody (4G9-1) in reaction zone 4,quetiapine antibody (11) in reaction zone 6, and risperidone antibody(5-9) in reaction zone 8. Table 7 below shows the results, confirmingthat there is no dose response except to the olanzapine antibody 4G9-1(in reaction zone 4).

TABLE 7 OLAN-Clone 4G9-1-Math Model 1 (0 ng/mL Conc.) Read Peak PeakMean Assay- Reaction Posi- Mean Mean Back- MM Conj Zone tion Area Heightground OLAN- ARIP, OLAN, 2 0.02 0.08 4.86 MM1 QUET, RISP OLAN- ARIP,OLAN, OLAN 4 34.23 51.80 5.39 MM1 QUET, RISP OLAN- ARIP, OLAN, 6 0.220.32 5.39 MM1 QUET, RISP OLAN- ARIP, OLAN, 8 0.15 0.17 5.59 MM1 QUET,RISP Only the Olanzapine Reaction Zone is binding

Referring to Table 8, a sample containing no quetiapine is deposited inthe sample zone and moves by capillary action through the conjugate zone(this time containing labeled quetiapine) and to the reaction zone. Thereaction zone again contains aripiprazole antibody (5C7) in reactionzone 2, as well as olanzapine antibody (4G9-1) in reaction zone 4,quetiapine antibody (11) in reaction zone 6, and risperidone antibody(5-9) in reaction zone 8. Table 8 below shows the results, confirmingthat there is no dose response except to the quetiapine antibody 11 (inreaction zone 6).

TABLE 8 Quetiapine-Clone 11-Math Model 1 (0 ng/mL Conc.) Read Peak PeakMean Assay- Reaction Posi- Mean Mean Back- MM Conj Zone tion Area Heightground QUET- ARIP, OLAN, 2 0.13 0.41 10.02 MM1 QUET, RISP QUET- ARIP,OLAN, 4 0.08 0.23 10.47 MM1 QUET, RISP QUET- ARIP, OLAN, QUET 6 140.35181.33 7.91 MM1 QUET, RISP QUET- ARIP, OLAN, 8 1.58 2.61 11.53 MM1 QUET,RISP Only the Quetiapine Reaction Zone is binding

Referring to Table 9, a sample containing no risperidone is deposited inthe sample zone and moves by capillary action through the conjugate zone(this time containing labeled risperidone) and to the reaction zone. Thereaction zone again contains aripiprazole antibody (5C7) in reactionzone 2, as well as olanzapine antibody (4G9-1) in reaction zone 4,quetiapine antibody (11) in reaction zone 6, and risperidone antibody(5-9) in reaction zone 8. Table 9 below shows the results, confirmingthat there is no dose response except to the risperidone antibody 5-9(in reaction zone 8).

TABLE 9 Risperidone-Clone 5-9-Math Model 1 (0 ng/mL Conc.) Read PeakPeak Mean Assay- Reaction Posi- Mean Mean Back- MM Conj Zone tion AreaHeight ground RISP- ARIP, OLAN, 2 1.03 1.51 9.07 MM1 QUET, RISP RISP-ARIP, OLAN, 4 0.65 0.91 9.60 MM1 QUET, RISP RISP- ARIP, OLAN, 6 2.616.39 10.48 MM1 QUET, RISP RISP- ARIP, OLAN, RISP 8 55.98 100.91 11.58MM1 QUET, RISP Only the Resperidone Reaction Zone is binding

The results shown above confirm that conjugates of labeled competitivebinding partners bind only to their respective antibodies in thereaction zone.

FIGS. 9-12 show typical dose response curves in specific antibodyreaction zones, and proof of dose response low/high concentration foreach specific assay in the presence of other conjugates. In FIG. 9, asample containing aripiprazole is deposited in the sample zone and movesby capillary action through the conjugate zone (this time containinglabeled aripiprazole, labeled olanzapine, labeled quetiapine, andlabeled risperidone) and to the reaction zone. The reaction zone againcontains aripiprazole antibody (5C7) in reaction zone 2. A typical doseresponse curve was generated as is shown in FIG. 9 only foraripiprazole, and not for olanzapine, quetiapine, or risperidone.

In FIG. 10, a sample containing olanzapine is deposited in the samplezone and moves by capillary action through the conjugate zone (this timecontaining labeled aripiprazole, labeled olanzapine, labeled quetiapine,and labeled risperidone) and to the reaction zone. The reaction zoneagain contains olanzapine antibody (4G9-1) in reaction zone 4. A typicaldose response curve was generated as is shown in FIG. 10 only forolanzapine, and not for aripiprazole, quetiapine, or risperidone.

In FIG. 11, a sample containing quetiapine is deposited in the samplezone and moves by capillary action through the conjugate zone (this timecontaining labeled aripiprazole, labeled olanzapine, labeled quetiapine,and labeled risperidone) and to the reaction zone. The reaction zoneagain contains quetiapine antibody (11) in reaction zone 6. A typicaldose response curve was generated as is shown in FIG. 10 only forquetiapine, and not for aripiprazole, olanzapine, or risperidone.

In FIG. 12, a sample containing risperidone is deposited in the samplezone and moves by capillary action through the conjugate zone (this timecontaining labeled aripiprazole, labeled olanzapine, labeled quetiapine,and labeled risperidone) and to the reaction zone. The reaction zoneagain contains risperidone antibody (5-9) in reaction zone 8. A typicaldose response curve was generated as is shown in FIG. 12 only forrisperidone, and not for aripiprazole, olanzapine, or quetiapine.

FIGS. 13-16 show typical dose response curves for each assay in thepresence of other conjugates and antibodies. In FIG. 13, a samplecontaining aripiprazole is deposited in the sample zone and moves bycapillary action through the conjugate zone (again containing labeledaripiprazole, labeled olanzapine, labeled quetiapine, and labeledrisperidone) and to the reaction zone. The reaction zone again containsaripiprazole antibody (5C7) in reaction zone 2, as well as olanzapineantibody (4G9-1) in reaction zone 4, quetiapine antibody (11) inreaction zone 6, and risperidone antibody (5-9) in reaction zone 8. Atypical dose response curve was generated for aripiprazole, as is shownin FIG. 13. When a sample containing olanzapine was deposited in thesample zone of this chip, a typical dose response curve was generatedfor olanzapine as shown in FIG. 14. When a sample containing quetiapinewas deposited in the sample zone of this chip, a typical dose responsecurve for quetiapine was generated as shown in FIG. 15. When a samplecontaining risperidone was deposited in the sample zone of this chip, atypical dose response curve for risperidone was generated as shown inFIG. 16.

FIGS. 17-20 show comparisons of dose response curves generated aspositive controls (FIGS. 5-8) to dose response curves generated in themultiplex format (FIGS. 13-16). The comparison for aripiprazole is shownin FIG. 17; for olanzapine in FIG. 18; for quetiapine in FIG. 19; andfor risperidone in FIG. 20. These figures show that the positive controlcurves are similar to the multiplex curves.

These data show that a lateral flow assay device of the subjectinvention can be used to detect multiple anti-psychotic drugs using asingle sample from a patient on one portable, point-of-care device.

What is claimed is:
 1. An isolated antibody or a binding fragmentthereof, which binds to aripiprazole comprising: a) a light chainvariable region comprising CDR1, CDR2 and CDR3 of SEQ ID NO:43, and aheavy chain variable region comprising CDR1, CDR2 and CDR3 of SEQ IDNO:44, wherein the light chain CDR1 sequence comprises amino acidresidues 46 to 55 of SEQ ID NO:43; the light chain CDR2 sequencecomprises amino acid residues 71 to 77 of SEQ ID NO:43; the light chainCDR3 sequence comprises amino acid residues 110 to 118 of SEQ ID NO:43;the heavy chain CDR1 sequence comprises amino acid residues 45 to 54 ofSEQ ID NO:44; the heavy chain CDR2 sequence comprises amino acidresidues 69 to 85 of SEQ ID NO:44; and the heavy chain CDR3 sequencecomprises amino acid residues 118 to 125 of SEQ ID NO:44; b) a lightchain variable region comprising CDR1, CDR2 and CDR3 of SEQ ID NO:47,and a heavy chain variable region comprising CDR1, CDR2 and CDR3 of SEQID NO:48, wherein the light chain CDR1 sequence comprises amino acidresidues 44 to 54 of SEQ ID NO:47; the light chain CDR2 sequencecomprises amino acid residues 70 to 76 of SEQ ID NO:47; the light chainCDR3 sequence comprises amino acid residues 109 to 117 of SEQ ID NO:47;the heavy chain CDR1 sequence comprises amino acid residues 45 to 54 ofSEQ ID NO:48; the heavy chain CDR2 sequence comprises amino acidresidues 69 to 85 of SEQ ID NO:48; and the heavy chain CDR3 sequencecomprises amino acid residues 118 to 126 of SEQ ID NO:48; c) a lightchain variable region comprising CDR1, CDR2 and CDR3 of SEQ ID NO:51,and a heavy chain variable region comprising CDR1, CDR2 and CDR3 of SEQID NO:52, wherein the light chain CDR1 sequence comprises amino acidresidues 44 to 58 of SEQ ID NO:51; the light chain CDR2 sequencecomprises amino acid residues 74 to 80 of SEQ ID NO:51; the light chainCDR3 sequence comprises amino acid residues 113 to 121 of SEQ ID NO:51;the heavy chain CDR1 sequence comprises amino acid residues 45 to 54 ofSEQ ID NO:52; the heavy chain CDR2 sequence comprises amino acidresidues 69 to 85 of SEQ ID NO:52; and the heavy chain CDR3 sequencecomprises amino acid residues 118 to 130 of SEQ ID NO:52; or d) a lightchain variable region comprising CDR1, CDR2 and CDR3 of SEQ ID NO:55,and a heavy chain variable region comprising CDR1, CDR2 and CDR3 of SEQID NO:56, wherein the light chain CDR1 sequence comprises amino acidresidues 44 to 54 of SEQ ID NO:55; the light chain CDR2 sequencecomprises amino acid residues 70 to 76 of SEQ ID NO:55; the light chainCDR3 sequence comprises amino acid residues 109 to 117 of SEQ ID NO:55;the heavy chain CDR1 sequence comprises amino acid residues 45 to 54 ofSEQ ID NO:56; the heavy chain CDR2 sequence comprises amino acidresidues 69 to 85 of SEQ ID NO:56; and the heavy chain CDR3 sequencecomprises amino acid residues 118 to 122 of SEQ ID NO:56.
 2. Theisolated antibody or a binding fragment thereof of claim 1, wherein theantibody fragment is selected from the group of fragments consisting ofFv, F(ab′), F(ab′)₂, scFv, minibody and diabody fragments.
 3. Theisolated antibody of claim 1, wherein the antibody is a monoclonalantibody.
 4. An assay kit comprising the isolated antibody or a bindingfragment thereof of claim
 1. 5. An assay device comprising the isolatedantibody or a binding fragment thereof of claim
 1. 6. The assay deviceof claim 5, wherein the device is a lateral flow assay device.
 7. Amethod of detecting aripiprazole in a sample, the method comprising: (i)contacting a sample with the antibody or binding fragment thereof ofclaim 1 labeled with a detectable marker, wherein the labeled antibodyor a binding fragment thereof and aripiprazole present in the sampleform a labeled complex; and (ii) detecting the labeled complex, therebydetecting aripiprazole in the sample.
 8. A competitive immunoassaymethod for detecting aripiprazole in a sample, the method comprising:(i) contacting a sample with the antibody or binding fragment thereof ofclaim 1, and with aripiprazole or a competitive binding partner ofpaliperidone, wherein one of the antibody or binding fragment thereofand the aripiprazole or competitive binding partner thereof is labeledwith a detectable marker, and wherein sample aripiprazole competes withthe aripiprazole or competitive binding partner thereof for binding theantibody or binding fragment thereof to form a complex; and (ii)detecting the amount of label to detect sample paliperidone.
 9. Themethod of claim 8, wherein the aripiprazole or competitive bindingpartner thereof is labeled with the detectable marker.
 10. The method ofclaim 8, wherein the antibody or binding fragment thereof is labeledwith a detectable marker.
 11. The method of claim 8, wherein theimmunoassay is performed on a lateral flow assay device and the sampleis applied to the device.
 12. The method of claim 7 or 8, wherein thedetection of aripiprazole is an indication of patient adherence withprescribed aripiprazole therapy.
 13. The method of claim 7 or 8, whereinthe detection of aripiprazole is used to determine whether a patientshould be converted from an oral aripiprazole regimen to an injectablearipiprazole regimen.
 14. The method of claim 7 or 8, wherein thedetection of aripiprazole is used to determine if the dose level ordosing interval of oral or injectable aripiprazole should be increasedor decreased to ensure attainment or maintenance of efficacious or safedrug levels.
 15. The method of claim 7 or 8, wherein the detection ofaripiprazole is an aid in the initiation of aripiprazole therapy byproviding evidence of the attainment of minimum pK levels.
 16. Themethod of claim 7 or 8, wherein the detection of aripiprazole is used todetermine bioequivalence of aripiprazole in multiple formulations orfrom multiple sources.
 17. The method of claim 7 or 8, wherein thedetection of aripiprazole is used to assess the impact of polypharmacyand potential drug-drug interactions.
 18. The method of claim 7 or 8,wherein the detection of aripiprazole is an indication that a patientshould be excluded from or included into a clinical trial and is an aidin the subsequent monitoring of adherence to clinical trial medicationrequirements.